Biological mechanisms in vivo are extremely complicated cascades of signals, which are difficult to deconvolute and understand. An example of such signalling is that required to activate B-cells. The B cell antigen receptor (BCR) is composed of membrane immunoglobulin (mIg) molecules and associated Igα/Igβ (CD79a/CD79b) heterodimers (α/β). The mIg subunits bind antigen, resulting in receptor aggregation, while the α/β subunits transduce signals to the cell interior. BCR aggregation rapidly activates the Src family kinases Lyn, Blk, and Fyn as well as the Syk and Btk tyrosine kinases. This initiates the formation of a ‘signalosome’ composed of the BCR, the aforementioned tyrosine kinases, adaptor proteins such as CD19 and BLNK, and signaling enzymes such as PLCγ2, PI3K, and Vav.
Signals emanating from the signalosome activate multiple signaling cascades that involve kinases, GTPases, and transcription factors. This results in changes in cell metabolism, gene expression, and cytoskeletal organization. The complexity of BCR signaling permits many distinct outcomes, including survival, tolerance (anergy) or apoptosis, proliferation, and differentiation into antibody-producing cells or memory B cells. The outcome of the response is determined by the maturation state of the cell, the nature of the antigen, the magnitude and duration of BCR signaling, and signals from other receptors such as CD40, the IL-21 receptor, and BAFF-R. Many other transmembrane proteins, some of which are receptors, modulate specific elements of BCR signaling. A few of these, including CD45, CD19, CD22, PIR-B, and FcγRIIB1 (CD32). The magnitude and duration of BCR signaling are limited by negative feedback loops including those involving the Lyn/CD22/SHP-1 pathway, the Cbp/Csk pathway, SHIP, Cbl, Dok-1, Dok-3, FcγRIIB1, PIR-B, and internalization of the BCR. In vivo, B cells are often activated by antigen-presenting cells that capture antigens and display them on their cell surface. Activation of B cells by such membrane-associated antigens requires BCR-induced cytoskeletal reorganization.
Autoreactive B cells are responsible for the production of pathogenic autoantibodies which can either directly or indirectly cause or exacerbate autoimmune conditions. Depletion of CD20 positive B cells has been used to successfully treat a number of autoimmune conditions and thus established conclusively that B cells play an important role in causing or maintaining a number of autoimmune diseases. Although B cell depletion has been a successful therapeutic option evidence also exists that control of B cell growth and activation status can also be an effective way to modulate B cell function. Alternative strategies that do not deplete B cells and offer the flexibility of controlling B cells without long term suppression of B cell immunity which has been shown to be associated with some side effects would therefore be desirable. In addition not all B cell responses or activities are harmful and evidence suggests that maintenance of regulatory B cell populations can be protective. Such an approach should be effective in diseases which have abnormal B cell function caused by inappropriate or excessive BcR signalling. Examples include, but are not limited to, inflammation, autoimmunity and cancer. Of particular interest are diseases that either have a direct requirement for BcR signalling or require inhibition or stimulation of humoral immune responses.
Bispecific antibodies are widely expected to play a major role in the next generation of biotherapeutics (D. Holmes, Nature Rev Drug Disc November 2011:10; 798). They have the potential to deliver superior, long term, broad efficacy in a greater proportion of patients. This can be achieved by either co-engaging different antigens simultaneously within a common disease pathway, thereby reducing redundancy; or by targeting antigens from independent pathways to provide an additive or synergistic effect.
To date strategies to inhibit B cell function without deleting the B cell have focused on exploiting the natural mechanism of regulation by CD32b (FcgRIIB). These include bispecific antibodies to CD79b/CD32b (Veri et al., Arthritis & Rheumatism 2010 62 1933-1943), CD19/CD32b (Karnell et al., J. Immunol 2014 192 1480-1490) and an antibody to CD19 with an Fc with enhanced CD32b binding (Chu et al., Arthritis & Rheumatology 2014 66 1153-1164).
Co-ligation of Fc gamma receptor IIb (CD32b) with the B cell receptor occurs to naturally regulate signalling, in particular when antigen is bound to antibody in small immune complexes. CD32b then recruits the phophatases SHP-1 and SHIP-1 which antagonise BcR activation. Although this natural regulatory mechanism can control B cell function, disruption of CD32b function caused by variation in the protein sequence of CD32b can lead to autoimmune disease and this receptor can be down regulated in autoimmune disease—e.g. as in the case of SLE. Alternative ways of blocking B cell activity are thus desirable as they offer alternative, non-natural, ways of regulating BcR function. These alternative mechanisms are likely to be particularly important when natural mechanisms are dis-functional in the given disease.
Bispecific antibodies facilitate access to novel biology such as:                1) cross-linking receptors on a cell, if appropriate,        2) inducing cell mediated effects,        3) localizing a cytokine to a cell to regulate signaling or locally block cytokine function,        4) engaging multiple epitopes simultaneously to generate “new activity”, increase function or specificity, which may not be exhibited by a single monoclonal antibody or indeed mixtures of un-linked antibodies (‘poly-monoclonals’), including mixtures directed to different antigens.        
The present inventors have surprisingly found that by using a bispecific antibody to couple the BcR (CD79) to the negative regulatory molecule CD22, which would, under normal physiological conditions be excluded from the complex, BcR signalling can be inhibited. CD22 is responsible for regulating tonic signalling through the BcR in the absence of antigen binding. However, upon antigen binding CD22 is normally excluded from the BcR complex. By physically linking the BcR with CD22 signalling through use of a bispecific antibody the inventors have found that activation in B cells can be inhibited.
The present inventors have therefore identified a synergistic function for molecules which are at least bispecific for CD22 and CD79. This function seems to be detectable primarily when binding regions with the combination of specificities are provided in a bispecific (multispecific) format, as opposed to simply being provided as a mixture of, for example monoclonal antibodies or binding fragments thereof.
The multispecific molecules of the invention are therefore useful in controlling aberrant B cell functions associated with certain diseases such as autoimmunity and cancer.